Leishmania are obligate intracellular protozoan pathogens of humans. Within infected patients, various species of this organism inhabit and destroy macrophages within the skin or internal organs (i.e., spleen, liver and bone marrow). Thus, they cause either ulcerative, non-healing, disfiguring malignant skin lesions (e.g. L. mexicana) or degenerative and most often fatal visceral disease (e.g. L. donovani). According to World Health Organization estimates, these diseases afflict over 12 million patients annually in the Tropics and Neo-tropics worldwide. Our studies are aimed at defining the mechanisms involved in the pathophysiology of these organisms. In that regard, the basic cell, molecular and developmental biology of Leishmania and related trypanosomatid protozoa are investigated toward identifying and characterizing parasite molecules which are essential for the survival of these human pathogens. How these parasites are able to survive, access nutrients, multiply and differentiate within their insect vector and mammalian hosts are questions central to understanding the basic parasitic nature and evolutionary adaptations of these organisms. Since these parasites interact directly with their hosts, knowledge of the composition and functions of their surface membranes seems essential. To that end, unique parasite surface membrane enzymes and regulatory proteins are identified and biochemically characterized to determine their functional roles in the survival of these organisms. Further, the genes encoding such proteins are being isolated and characterized for the first time, toward defining their expression and regulation during course of parasite growth, differentiation and development. For example, the genes for several L. donovani tartrate-sensitive, histidine, secretory- (LdSAcPs) and a unique tartrate-resistant, surface membrane- (LdMAcP) acid phosphatases were used as probes in molecular studies. Results of combined biochemical and molecular studies showed that this family of enzymes was functionally conserved among all pathogenic leishmanial species examined as well as, other more distantly related trypanosomatid parasites of humans. The conservation of such LdSAcPs and LdMAcP homologs amongst all pathogenic Leishmania sps. suggests that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. In collaboration with P.A. Bates, we demonstrated that the unique trypanosomatid surface membrane enzyme, a 3'-nucleotidase/nuclease was conserved in Leishmania mexicana and that it was differentially and developmentally expressed in this parasite. Further, our molecular analyses showed that this leishmanial enzyme is unique being the only surface membrane-anchored member of the Class-I family of exo-nucleases. Our combined results of these studies have demonstrated both the ubiquity and structural conservation of the 3'NT/NU among all pathogenic Leishmania parasites. In parallel biochemical and molecular studies, we recently identified in a new thiol-sensitive, surface membrane, exo-ribonuclease in L. donovani. This finding provides yet another unique putatively essential surface membrane nucleoside-salvage enzyme as potential chemotherapeutic target in this pathogen. In other studies, we demonstrated that the L. donovani calreticulin (LdCal) protein functions as a lectin-like chaperone in facilitating the proper folding and processing of glycoproteins (e.g., the leishmanial secretory acid phosphatase) within the parasite's endoplasmic reticulum. Results obtained from the use of both anti-sense and over-expression constructs of the LdCal demonstrated that this chaperone-protein is essential for parasite growth and survival. Results of our recent studies continue to provide pertinent information toward understanding the unique pathophysiology of these organisms. In addition, these studies are of practical relevance toward demonstrating whether such parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.